Sulfur oxidation in selected human cortical cataracts and nuclear cataracts

Exp. Eye Res. (1980) 31, 361-369 Sulfur Oxidation in Selected Human Cortical Cataracts and Nuclear Cataracts MARGARET I t . GARNER AND ABRAHAM SPECTOI~

Biochemistry and Molecular Biology Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia Unive~'sity, New York, N.Y. 10032, U.S.A. (Received 8 January 1980, New York) The water soluble protein, the water insoluble non-membrane protein, and the membrane fraction were isolated from b o t h the cortex and nucleus of nuclear and cortical cataracts. Measurements of cysteine and methionine oxidation in these fractions indicated t h a t sulfur oxidation occurs in the transparent as welt as the opaque region of the lenses. Cysteic acid is found only in the opaque region. I n the nucleus of nuclear cataracts, extrinsic and cytoplasmic polypeptides are found eovalently linked b y disulfide bonds to the intrinsic membrane. I n the cortex of cortical cataracts, there is no intrinsic membrane involvement. However, a h i g h molecular weight disulfide linked aggregate can be isolated from the cortex of cortical cataracts. This aggregate appears to contain the same extrinsic and cytoplasmic proteins as are found covalently attached to the membrane of the nuclear cataracts. Key words: h u m a n ; cataract; oxidation; methionine; eysteine.

1. Introduction

The post-translational modification of amino acids in the proteins in senile cataract appears to involve oxidatioD of selected groups. The number of disulfide bonds (Anderson and Spector, 1978; Anderson, Wright and Spector, 1979) and the amount of methionine sulfoxide increases with the severity of the cataract (Truscott and Augusteyn, 1977). In the aging normal lens, cysteine and methionine oxidation occur only in the intrinsic membrane fraction and the high molecular weight (HMW) membrane associated fractions (Garner and Spector, 1980). In the severe eataractous lens, the oxidation of the sulfur amino acids is found not only in the intrinsic membrane fraction, but in all other fractions of the lens. Higher sulfur oxidation states such as cysteic acid and methionine sulfone are observed in membrane related fractions of these severe cataraetous lenses. The question then arises as to whether sulfur oxidation is present in the clear as well as opaque regions of eataractous lenses with localized opacities. This report describes a study of methionine and cysteine oxidation in the cortex and nucleus of selected sets of well defined nuclear and cortical cataracts. The data suggests that eysteine and methionine oxidation does occur in the clear region of these lenses. However, higher sulfur oxidation states, disulfide linked HMW aggregates, and cytoplasmic polypeptides disulfide linked to membrane are observed only in the opaque regions of these lenses. 2. Materials and Methods

Freshly obtained cataractous lenses were photographed (Chylack, 1978), classified and stored at --80 ~ prior to use. Decapsulated lenses were used in all studies. The lenses were separated into nucleus and cortex using a 6 mm trephine which yields 40% of the lens as cortex and 60% of the lens as nucleus. All chemicals used were reagent grade. 0014-4835/80/090361-t-09 $01.0010

9 1980 Academic Press Inc. (London) Limited 361

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M.H. GARNER AND A. SPECTOR

The cortex and nucleus from these lenses were homogenized in 0-1 M-KC1/0.03M-Tris/ 0.05 M-iodoacetamide pH 7"4. The water soluble (WS) protein was separated from the homogenate by centrifugation at 60 000 • g. The intrinsic membrane fraction was isolated from the pellet (Roy and Spector, 1978a). The pellet was suspended in 6 ~-guanidine hydrochloride and reacted with citraconic anhydride. Methyl iodide was added to block thiol and thioether groups to prevent oxidation and facilitate analysis. After the citraconylated material was dialyzed exhaustively against 0.2% ammonia, the material was centrifuged at 60 000 • The pellet was resuspended and spun at 60 000 • g until no proteiu could be detected in the supernatant. This pellet, the membrane fraction, was stored at 4~ prior to use. The combined supernatants, the water insoluble nonmembrane (WINM) protein, were dialyzed against deionized water and lyophilized. The procedure used to determine methionine and its oxidation products and eysteine and its oxidation products were described previously (Garner and. Spector, 1980). Methionine sulfone was determined directly by amino acid analysis. Methionine sulfoxide was obtained as methionine sulfone after performic acid oxidation of a methylated sample. Methionine was determined as methyl methionine by amino acid analysis of p-toluene sulfonic acid hydrolysis of the methylated protein. Cysteine was determined as either carboxymethyl or methyl cysteine and cystine as an increase of carboxymethyl eysteine following reduction with dithiothreitol and alkylation with iodoaeetamide. Cysteic acid was analyzed directly. Under the sensitivity conditions presently used with the amino acid analyzer, 0.1 of a residue of derivatized sulfur containing amino add can be detected. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed using the procedure of Fairbanks, Steck and Wallaeh (1971) or Roy and Spcctor (1978b, c). Amino terminal amino acid determinations were performed using potassium cyanate (Stark and Smythe, 1973), Weights of the various protein fractions were determined on lyophilized samples using the procedure of Garner and Speetor (1978). The percentage of bound water in these fractions was determined by amino acid analysis of a weighed sample of protein. Lipid content of the lens membrane fraction was taken into account in the weight determination for all membrane fractions. 3. Results

Stereophotographs of both the anterior and posterior views of representative cataracts in the two groups considered in this discussion are shown in Fig. 1. Figure l(a), (b) shows a representative nuclear cataract. The opacity is confined mainly to the nucleus of the lens. The outer 40% of the lens appears clear. Figure l(c), (d) show a representative cortical cataract. The opacity appears to be confined to the outer 30% of the lens. The opacity is more dense on the posterior side of the lens. There is also increased color in the nucleus of these lenses but there is no opacity. Older normal lenses also show some increased yellowing in the nuclear region (Said and Weale, 1959; Bando, Nakajima and Satoh, 1975; Lerman, Yamanashi, Palmer, Roark and Borkman, 1978). An examination of the oxidation of methionine and cysteine in the protein fractions isolated from a pool of seven nuclear cataract lenses are shown in Table I. In the nuclear region of these lenses, approximately 25% of the methionine is methionine sulfoxide in the water soluble and water insoluble fractions. The nuclear membrane contains no detectable methionine sulfoxide. This result is somewhat surprising since all other fractions contain methionine sulfoxide. Two separate sets of nuclear cataracts have been examined and both times there were no detectable oxidation products of methionine in the membrane fraction. In the nuclear WS fraction 48% of the eysteine is in disulfide linkage. The nuclear WINM fraction has 63% of the cystcine in the disulfide state. In the nuclear membrane 66% of the eysteine is in S-S linkages

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M. It. G A l ~ N E I ~ A N D A. S P E C T O R

and another 13% is cysteic acid. The WS fraction of the transparent cortex of the same lenses has 22~o of the methionine as methionine sulfoxide and 57~o of the cysteine in disulfide linkage. The cortical WINSI fl'aetion contains 18% of' its methionine as methionine su]foxide and 16% of the eysteine in disulfide bonds. In the intrinsic membrane fraction of the cortex of these nuclea,r cataracts 17% of the methioninc is present as methionine sulfoxide and 50~ of the cysteine as cystine. TABLE I

Oxidation of the sulfur amino acids in fractions from the cortex and nucleus of nuclear cataracts*

Methionine

Methionine sulfoxide

Cysteine

Cysteine in S-S

Cysteic acid

3'5 3'6 5'6

1-2 1.2 0

1.3 1.3 0.8

1.2 2-2 2-5

0 0 0.5

2.9 2.8 4.9

0-8 0-6 1.0

1.8 2.1 1.0

1.8 0.4 1.3

0 0 0

lYueleu8

WS protein t WINN: protein S Membrane Cortex

WS protein t W I N M protein:~ Membrane

* The data is expressed as residues/200 residues. One pool of seven lenses was used to obtain the present data; similar results were obtained with a second pool of lenses. Water soluble protein. Water insoluble non membrane protein, solubilized by modification of z-amino groups with eitraconio anhydride.

The oxidation state of methionine and cysteine in the protein fractions of the cortex and nucleus of the cortical cataracts are listed in Table II. Between the cortex and nucleus of these lenses, there appears to be little difference in the extent of oxidation of mettEonine. Comparable amounts of unoxidized cysteine are also found in the nuclear and cortical fractions. The major difference is in the quantity of eysteine disulfide and cysteic acid. In the cortex, the amount of cysteine in S S linkages is 32, 45 and 31% for the WS, WINM and membrane fractions respectively. Cysteic acid is found in both the WS protein and membrane fractions of the cortex. In the nucleus 50% of the cysteine of the WS and WINM protein fractions is in disulfide linkages; 71~o of the cysteine of the membrane fraction is in disulfide linkage. There is no detectable cysteie aeid in tile nucleus. In normM lenses of similar age no oxidation was observed in the non-membrane related fractions. However, in the membrane fraction from either the cortex or nucleus approximately 20yo of the methionine was in the sulfoxide state and approximately 50o/0 of the cysteine was in the disulfide form. In young normal lenses there is no oxidation detected in any lens fraction. The membrane isolated from the nuclear region of the nuclear cataracts has some unusual properties. First, 55yo of the total mass is associated with the membrane and consists of two fractions, a heavy yellow component and a lighter white fraction. Based on electron microscopy, both fractions contain membrane. A similar observation of two membrane fractions was reported in an earlier study of severe, totally opaque lenses (Garner and Spector, 1980). Second, the preparation does not migrate into

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365

SDS-PAGE gels (procedure of Fairbanks et al., 1971) when the samples are incubated without reduction. Third, the amino acid composition of the membrane fraction is unusual. In conjunction with the examination of the oxidation of methionine and cysteine, weights of the lyophilized protein fractions were determined using the procedure of Garner and Speetor (1979). Lyophilized weights were also determined for similar fractions isolated from the nucleus and cortex of normal lenses of approximately the same age. Bound water in the lyophilized samples constitutes 30-35~ of the weights determined above. This was ascertained for the WS and WINM fractions by comparison of lyophilized weights with weights determined by amino acid analyses. This level of residual H20 appears to be constant for all non-membrane fractions studied. TABL~

II

Oxidation of the sulfur amino acids in fractions from cortex and nucleus of cortical cataracts*

Methioninc

Methionine sulfoxide

Cysteine

Cysteine as S-S

Cysteic acid

3-5 3.2 4.2

0.5 0.5 0.8

1.5 1.1 0.8

0.8 0-9 0.6

0.2 0 0.5

3.6 3-4 4-4

0.4 0.6 0.6

1.5

1-5

0

1.1 0.8

1.1 2.0

0 0

Cortex

WS protein WINM protein Membrane .Nucleus

"

WS protein WINM protein Membrane

* The data is expressed as residues/200 residues. See notes below Table I for abbreviations and further information. TABLE III

Relative weight percent distribution among the fractions isolated from the nuclear and cortical regions of normal, cortical and nuclear cataract lenses*

Normal lens Normal lens nucleus cortex WS protein WINM protein Membrane

58i5 39• 3=~ 1

56• 37J=6 512

Cortical cataract nucleus

Cortical cataract cortex

Nuclear cataract nucleus

Nuclear cataract cortex

52~4 45~=4 3• 1

34• 62i5 3•

12i3 33~6 55i6

5314 44• 3-4-1

* See Table I for abbreviations.

In the case of normal membrane the protein to lipid ratio is 1 : 1. This value in conjunction w~ith amino acid analyses was used to check for bound water in the membrane fractions. It appeared to be 25-35% of the lyophilized weight. Therefore, the relative percentages of H20 are approximately the same as for the non-membrane fractions. The results of relative weight percent distribution calculated from these

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determinations are listed in Table III. In all cases, the values reported are the average from two separate lens preparations. In the nucleus of the nuclear cataract approximately 55% of the total weight is in the membrane fraction. In the transparent cortex of the nuclear cataracts and the transparent nucleus of the cortical cataracts, the percentages are similar to those found with normal lenses. In the cortex of the cortical cataract, there is an increase in the amount of WINM protein. The average total dry weight per lens was 73.], 77.3 and 70.2 mg for the normal, cortical cataract and nuclear cataract preparations respectively. These lenses appear to have lost no protein because of the opacity.

A

B

C

FIG. 2. SDS gel electrophoresis of nuclear membrane preparations from nuclear cataracts. A, PAGE profile of nuclear membrane without reduction; B, PAGE profile of nuclear membrane with mercaptoethanol; C, agarose, 0.5%, polyacrylamide, 2.5% gel electrophoresis profile of material released from the membrane by reduction and alkylation.

Typical SDS-PAGE patterns for the membrane from the nucleus of the nuclear cataract are shown in Fig~,2. In the gel where the sample was incubated in SDS, without mercaptoethanol; jn the presence of iodoaectamide, virtually none of the material enters the gel (Fig. 2A). In the gel containing samples incubated with mercaptoethanol, the typical membrane polypeptides are observed (Fig. 2B). However, considerable material remains at the top of the gel. Two major conclusions result from this experiment. First, there are clearly disulfide linkages which have immobilized the intrinsic membrane polypeptides in large aggregates. Second, even after reduction there is an appreciable amount of protein which does not penetrate the gel. The latter material which does not penetrate the Fairbanks gel appears to be released from the membrane. Therefore, the membrane preparation was reduced, alkylated, recitraeonylated, dialyzed against NHdOH and centrifuged. The supernatant was collected, lyophilized and run on SDS polyacrylamide/agarose gels (Roy and Spector, 1978). The pattern is shown in Fig. 2(@ A minor 43 000 dalton band, a 24 000 dalton component, a low molecular weight band as well as a streaky diffuse region can b e observed. This material does not penetrate Weber Osborn gels as was reported earlier for a reduced high molecular weight disulfide linked aggregate (Roy and Spector, 1978).

OXIDATION

367

IN HUMAN LENSES

The amino acid composition of the nonreduced nuclear cataract membrane fraction is listed in column 1, Table IV. For comparative piirlJoses the composition of normal membrane is listed in column 2. (No difference is observed between membrane isolated from cortex and nucleus of normal lenses). The samples, prior to hydrolysis, were reduced and alkylated to aid in the quantitation of eysteine. Because of this procedure eysteine is reported as earboxymethyl eysteine. The membrane of the nucleus of the nuclear cataracts has more polar amino acids and fewer nonpo!ar TABL~ IV

Comparison of amino acid compositions of membrane preparations* Treated nucIear Nuclear cataract NormaI lens membrane from nuclear membrane membrane nuclear eataraett Carboxymethyl cysteine Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleueine Leueine Tyrosine Phenylalanine Lysine tIistidine Arginine

4 . 18 8 14 24 14 16 13 10 5 10 16 13 11 7 5 16

2 .

. 11 10 14 14 l0 21 22 15 5 7 25 8 14 5 5 11

Protein released

2

4

13 8 12 20 11 20 18 lI 5 8 23 9 13 5 5 11

18 6 13 25 12 17 9 12 5 10 16 14 I1 7 5 15

.

24 000 dalton polypeptide,+ -3w 18 7 12 27 15 16 10 12 5 10 15 13 1I 9 5 16

* Expressed as residues/200 residues. t The membrane preparation was reduced, alkylated, reeitraeonylated, dialyzed and repelleted. :~ The polypeptide has been isolated and characterized in this laboratory by Dr. W. H. Garner and shown to be a y-erystallin. w Cysteine value may be low because the composition was determined on protein that had not been treated with a reducing agent and iodoaeetamide.

amino acids than its normal counterpart. This dramatic change in composition is caused by the additional protein in this fraction. There is also more cysteine in the cataract membrane fraction. Aliquots of the pellet of the reduced, alkylated and recitraeonylated membrane and material released into the supernatant by reduction were hydrolyzed and taken for amino acid analysis. The compositions of the membrane (the pellet) and the supernatant are listed in columns 3 and 4 of Table IV. Although there is not complete identity, the membrane composition compares more closely with that of the normal lens. The released material had a composition very similar to a ?-crystallin polypeptide isolated from human water soluble protein. Using the cyanate procedure to determine the amino acid terminal groups in the fraction, 90~o of the protein in the fraction had a free glycine terminal. The supernatant also showed weak reactivity with a calf ~,-crystallin antiserum. The cortical membrane of the nuclear cataract and the nuclear and cortical membrane of the cortical cataract showed very little evidence of this type of membranecytoplasmic protein aggregate.

368

M.H. G A R N E R AND A. S P E C T O R

4. Discussion

The results obtained in the study of nuclear and cortical cataracts indicate that sulfur oxidation is present in both the clear and the opaque regions of the lens. In the nuclear cataracts, the water insoluble non-membrane fraction and the isolated membrane fraction of the opaque region were more oxidized than similar fractions isolated from the clear cortical region. In the cortical cataract group approximately the same level of oxidation was observed in the transparent and opaque region. However, in both sets of lenses, cysteic acid was observed only in the fractions isolated from the opaque region. Mixed disulfides with glutathione were not observed in this study. This does not necessarily mean that no mixed disulfides are present. In the earlier study, (Garner and Spector, 1980), evidence for mixed disulfides were found only in the membrane and disulfide linked high molecular weight fraction of severe cataract lenses. The intrinsic membrane was found to be linked to cytoplasmic and extrinsic membrane components in the nucleus of the nuclear cataract." The non-intrinsic membrane polypeptides involved appear to be the same as those reported in an earlier study (Garner and Spector, 1980), mainly a 24 000 dalton polypeptide related to y-crystallin, the extrinsic membrane 43 000 dalton polypeptide and a heterogeneous low molecular weight fraction (8500-11 000 dalton). The latter fraction isolated from the water soluble and water insoluble components of cataractous lenses have previously been partially characterized (Roy and Spector, 1978b; Garner, Garner and Spector, 1979). The amino acid composition of this fraction calculated per 200 residues instead of 10 000 daltons is very similar to the human ?-crystallin composition. The low molecular weight population was shown to be quite heterogeneous reacting with antisera to ~-, fi- and y-crystallin as well as an antiserum to the 43 000 dalton polypeptide. Disulfide linkage of non-intrinsic membrane polypeptides to the membrane was not observed in either the cortex or nucleus of the cortical cataracts used for this study. Preliminary data on these cortical cataracts indicate the presence of large disulfide linked high molecular weight aggregates in the cortex which can be separated from the membrane without reduction or alkylation. Such aggregates were not observed in the nuclear cataract. Thus the major difference between clear and opaque regions of either nuclear or cortical cataracts appears to be the level of oxidation and the presence of disulfide linked aggregates involving either extrinsic and cytoplasmic species or intrinsic, extrinsic and cytoplasmic species. The fact that the two classes of cataract studied appear to have some different properties raises a question concerning the mechanism of oxidation, disulfide formation and membrane involvement. The intrinsic polypeptides do not appear to be significantly involved in disulfide linkage with cytoplasmic polypep~ides in developing cortical cataracts but are involved in nuclear cataract and cataracts involving the entire lens. Such observations suggest a reairangement in the architecture of the membrane and its underlying matrix resulting in the exposure of the intrinsic polypeptides and the formation of disulfide linkage with other polypeptide species. It is of interest to note that the present data suggest that the same nonintrinsic polypeptides are involved in both cortical and nuclear cataract.

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ACKNOWLEDGMENTS The skilled technical assistance of Gloria Jenkins and Olivia Lovelaee is gratefully acknowledged. This work was supported by grants from the National Eye Institute, National Institutes of Health, Department of Health, Education and Welfare. Dr M. H. Garner is a National Research Service Award recipient. REFERENCES

Anderson, E. I. and Spector, A. (1978). The state of sulfhydryl groups in normal and cataractous human lens proteins. I. Nuclear region. Exp. Eye Res. 26~ 407-17. Anderson, E. I., Wright, D. D. and Speetor, A. (1979). The state of sulfhydryl groups in normal and cataractous human lens proteins. II. Cortical and nuclear regions. Exp. Eye Res. 29~ 233-43. :Bando, M., Nakajima, A. and Satoh, K. (1975). Coloration of human lens protein. Exp. Eye Res. 20, 489-92. Chylack, L. T. (1978). Classification of human cataracts. Arch. Ophthalmol. 96, 888-92. Fairbanks, G., Steck, T. L. and Wallach, D. F. H. (1971). Electrophoretic analyses of the major polypeptides of the human erythrocyte membrane. Biochemistry 13, 2606-17. Garner, W. H., Garner, M. H. and Spector, A. (1979). Comparison of the 10 000 and 43 000 dalton polypeptide populations isolated from the water soluble and insoluble fractions of human cataractous lenses. Exp. Eye Res. 29, 257-76. Garner, M. H. and Spector, A. (1980). Selective oxidation of cysteine and methionine in normal and cataractous lenses. Proc. Natl. Acad. Sci., U.S.A. 77, 1274-77. Roy, D. and Spector, A. (1978). Human insoluble lens protein. I. Separation and partial characterization of polypeptides. Exp. Eye Res. 26, 42943. Roy, D. and Spector, A. (1978). Human insoluble lens protein. II. Isolation and characterization of a 9600 dalton polypeptide. Exp. Eye Res. 26, 445-59. Roy, D. and Spector, A. (1978). Disulfide-linked high molecular weight protein associated with human cataract. Proc. Natl. Acad. Sci., U.S.A. 75, 3244-8. Said, F. S. and Weale, 1~. A. (1959). The variation with age of the spectral transmissivity of the living human crystalline lens. Gerontologia3, 213. Stark, G. R. and Smythe, D. G. (1963). The use of cyanate for the determination of NH2-terminal residues in protein. J. Biol. Chem. 238, 214-26. Trnscott, 1~. J. W. and Augusteyn, R. C. (1977). Oxidative changes in human lens proteins during senile nuclear cataract formation. Biochim. Biophys. Aeta 492, 43-52.